ProjectSummary?Project3 HSP70isakeycancer-criticalsurvivalproteinthatisnecessarytomaintainproperproteinfoldinginastressed cell.TheHSP70familycontainsmorethaneightfamilymembers.Theoneunderstudyhereisthemajorstress- inducedfamilymemberHSPA1A,hereafterHSP70.Unlikeotherfamilymembers,thisproteinisoverexpressed inover75%ofmetastaticmelanoma,andisassociatedwithdrugresistanceandpoorsurvival.Inthepastten yearswehavedevelopedaseriesofinhibitorsthattargetthestress-inducedformofHSP70butnototherfamily memberslikeHsc70andGRP75.Additionally,werecentlynotedasignificantfractionofHSP70locatedatthe mitochondriaoftumorbutnotnormalcells,andwecoupledourinhibitorstoatriphenylphosphoniummoietythat helps direct these compounds to mitochondria. In the past funding cycle we tested this compound against melanoma, and solved the crystal structure and mechanism of action. In the current funding cycle, we take newerandmorepotentderivativesofournovelmitochondria-directedHSP70inhibitorstothetoughest-to-treat subtypes of melanoma:thosethatare resistant to BRAF/MEK inhibitors, those that have wild type BRAFand NRAS (WT/WT), and those that have metastasized to the brain (melanoma brain metastases, or MBMs). Targetingthesethreecategorieshasthepotentialforsignificantclinicalimpact,andourpreliminarydatasupport the use of HSP70i for these tumor sub-types. In the proposed work we collaborate extensively with our P01 colleaguestoexploreforthefirsttimetheimpactofHSP70ionthestressedtumormicroenvironment,including cancerassociatedfibroblastsand theaged micro-environment. We alsofocuson several new HSP70 clients, including ErbB3,ID3 and GPX4. To succeed in thesegoals, we haveaccrued a team that is expert in HSP70 and proteostasis (Murphy and George), medicinal chemistry (Salvino) and the brain metastatic micro- environment(Chen).WeexpecttheproposedresearchtoyieldcandidateHSP70iforclinicaldevelopment,and toprovideanimportanttherapeuticoptionforthetoughest-to-treatmelanomasub-types.